Rapid characterization of HIV-1 sequence diversity using denaturing gel electrophoresis and direct automated DNA sequencing of PCR products

Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
PCR methods and applications 06/1993; 2(4):293-300. DOI: 10.1101/gr.2.4.293
Source: PubMed


A direct method for visualization and isolation of sequence variants of human immunodeficiency virus type 1 (HIV-1) utilizing denaturing gradient gel electrophoresis (DGGE) combined with automated direct DNA sequencing was developed. Two fragments from the env gene and one from the nef gene of HIV-1, which together constitute approximately 1.0 kb of sequence, were amplified by PCR and analyzed. HIV-1 variants from each region were resolved and excised from the gel; this was followed by direct sequencing of different viral variants. In 9 infected patients, a limited number of dominant sequence variants could be seen in the three regions, together with a faint background of minor variants. The use of DGGE makes it possible to obtain a direct estimate of overall HIV-1 sequence diversity within patient samples without an intermediate DNA cloning step.

Download full-text


Available from: Bjorn Andersson,
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: High mutation rates and strong selective pressures imposed on human immunodeficiency viruses in vivo result in the formation of pools of genetic variants known as quasispecies. DNA heteroduplex mobility and tracking analyses were used to monitor the generation of HIV sequence diversity, to estimate quasispecies complexity, and to assess the turnover of genetic variants to approach an understanding of the relationship between viral quasispecies evolution in vivo and disease progression. Proviral DNA pools were nearly homogeneous soon after sexual transmission. The emergence and clearance of individual variants then occurred at different rates in different individuals. High quasispecies complexity was found in long-term-infected, asymptomatic individuals, while rapid CD4+ cell decline and AIDS were often, but not always, associated with lower quasispecies complexity. Proviral genetic variation was often low following in vitro culture, because of the outgrowth of one or a few variants that often became more abundant only later as proviruses in peripheral blood mononuclear cells. These studies provide insight into the dynamics of human immunodeficiency virus sequence changes in vivo and illustrate the utility of heteroduplex analysis for the study of phenomena associated with rapid genetic changes.
    Journal of Virology 11/1994; 68(10):6672-83. · 4.44 Impact Factor

  • PCR Strategies, 01/1995: pages 154-160; , ISBN: 9780123721822
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fifty clinical strains of Chlamydia trachomatis were studied by denaturing gradient gel electrophoresis (DGGE) of bacterial DNA amplified by the polymerase chain reaction (PCR). The strains belonged to the three most commonly encountered serovars in developed countries--D, E and F. Six reference strains, including the serovar Da strain, were also studied. The DNA sequences explored encompassed the four variable domains (VDs) of omp1, the gene encoding the major outer-membrane protein (MOMP). The corresponding regions in the MOMP contain the species-, subspecies- and serovar-specific epitopes. The four distinct serovars were clearly differentiated by specific migration pattern. No sequence variations were observed among strains of serovar F. However, variant strains within serovars D and E were found, which exhibited migration patterns different from those of the reference strains and these were sequenced directly. According to the observed sequence variations, serovar D strains could be divided into three stable representative groups (D, D1 and D2). Two variants were identified among serovar E strains. No biological differences were observed for the variant strains in terms of growth properties, ecology or pathogenicity. All the nucleotide substitutions detected in the VDs were non-synonymous at the protein level and, for the serovar D strains, could account for differences identified by specific monoclonal antibodies. These substitutions could be involved in antigenic drift, driven by the immune pressure of the host, leading to the emergence of serovariants. The data may explain, in part, chlamydial infection recurrences and could have critical implications for developing rational vaccine strategies.
    Journal of Medical Microbiology 08/1995; 43(1):14-25. DOI:10.1099/00222615-43-1-14 · 2.25 Impact Factor
Show more